Prostate specific antigen (PSA) was first described by Wang, M. C., et al., Invest Urol, 17:159 (1979). It is a secretion of prostate epithelium and is also produced by prostate cancer cells. PSA was characterized as a glycoprotein monomer of 33-34,000 dalton molecular weight with protease activity (Wang, M. C., et al., supra and Ban, Y, et al., Biochem Biophys Res Commun. 123:482 (1984)). More recently the amino acid sequence of the antigen has been reported (Watt, W. K., et al., Proc Natl Acad Sci USA, 83:3166 (1986)) and the gene for PSA has been cloned (Lundwall, A., Biochem Biophys Res Commun. 161:1151 (1989)). Development of an enzyme immunoassay by Kuriyama, et al, made it possible to detect low concentrations of PSA in the blood of patients with malignant and benign prostate disease and a significant proportion of normal males (Kuriyama, M. et al., Cancer Res. 40:4658 (1980)).
PSA testing can have significant value in detecting metastatic or persistent disease in patients following surgical or medical treatment of prostate cancer (Lange, P. H., et al., Urology 33(6 Suppl): 13, (June, 1989); and Killian, C. S., et al., Cancer Res 45:886 (1985)). Persistent elevation of PSA following treatment or an increase in the post-treatment baseline PSA level is indicative of recurrent or residual disease (Brawer, M. K., et al., Urology, 33 (5 Suppl): 11 (May, 1989); Siddal, J. K., et al., Eur Uro12:1:3 (1986); Stamey, T. A., et al., N Engl J Med 317:909 (1987); Lange, P. H. et al., J Urol. 141:873 (1989); Stamey, T. A., et al., J Urol. 141:1076 (1989); Stamey, T. A., et al., J Urol 141:1084, (1989); Stamey, T. A., et al., J Urol 141:1088 (1989); and Chan, D. W., et al., Clin Chem 33:1916 (1987)).
PSA testing alone is not recommended as a screening procedure in the general population nor as a guide in disease staging. Instead, it is widely accepted as an adjunctive test in the management of prostate cancer patients (Kuriyama, M. et al., J Natl Cancer Inst 68:99 (1982 ); Stamey, T. A., et al., N Engl J Med 317:909 (1987); Lange, P. H. et al., J Urol. 141:873 (1989); Stamey, T. A., et al., J Urol. 141:1076 (1989); Stamey, T. A., et al., J Urol 141:1084 (1989); Stamey, T. A., et al., J Urol 141:1088 (1989); Chan, D. W., et al., Clin Chem 33:1916 (1987); and Oesterling, J. E., et al., J Urol. 139:766 (1988)).
Measurements of the serum concentration of PSA have now found widespread use in monitoring of patients with prostate cancer, although increased serum concentrations of PSA have also been reported in benign prostatic hyperplasia and secondary to surgical trauma of the prostate (Duffy, Ann Clin Biochem, (1989); Brawer, et al., Urology Suppl, (1989)).
PCT patent application WO 92/01936, "Assay of Free and Complexed Prostate Specific-Antigen (PSA)" to Lilja, H. et al., published Feb. 6, 1992, discloses immunoassays for free PSA as well as PSA as a proteinase inhibitor complex. The free PSA and the PSA complex are measured by a non-competitive immunoassay employing at least two different monoclonal antibodies. The invention is further characterized in that the PSA proteinase inhibitor complex of interest is formed either with .alpha..sub.1 -antichymotrypsin (ACT), .alpha..sub.1 -protease inhibitor (API) or .alpha..sub.2 -macroglobulin. Moreover, the invention is characterized in that free PSA, the PSA-proteinase inhibitor complex and their ratio to total PSA are applied in the diagnosis of patients with prostate cancer.
The patent application discloses three monoclonal antibodies ("MAB"). PSA complexed to ACT ("PSA-ACT complex") and free PSA were identified by MAB 2E9 and 2H11. MAB 5A 10 recognizes free PSA but not PSA-ACT complex. MAB 2E9 is the only anti-PSA MAB that readily identified free PSA and PSA-ACT complex on immunoblots. None of the anti-PSA MAB 2E9, 2H11 or the 5A10 significantly blocked the binding of each other to solid phase-bound PSA.
By using different combinations of the MAB in non-competitive immunoassays of human sera, the application found that increased clinical specificity is achieved by measuring both free PSA and PSA-ACT complex and that the ratios between free PSA/total PSA and free PSA/PSA-ACT complex are significantly different between benign prostatic hyperplasia and prostate cancer patients.
MAB specific for free PSA and those reactive with PSA-ACT complex are commercially available, for example, from CanAg Diagnostics AB, Gothenburg, Sweden. Through inhibition studies and analyses of dose-response of different combinations of their MAB, CanAg Diagnostics AB found at least 9 major antigenic determinant groups on the PSA molecule. One group of its MAB-defined epitopes was exposed both on uncomplexed PSA and PSA-ACT complex, and another group of its MAB-defined epitopes was exposed only in free PSA. (CanAg Diagnostics AB, Nilsson et al., Epitope mapping of PSA, and development of assays for determination of different isoforms of PSA, and the abstract of the same title, Abstract P 38, J. Tumor Marker Oncology, 10th Int'l Conference on Human Tumor Markers, Sep. 8-11, 1993, Bonn, Germany).
Competitive radioimmunoassays for PSA are commercially available (e.g., from PROS-CHECK PSA, Yang Laboratories, Inc., Bellevue, Wash.). PROS-CHECK PSA uses polyclonal rabbit antibodies to PSA, and PSA labelled with Iodine.sup.125.
Currently, there are two types of PSA non-competitive sandwich immunoassays on the market. The first type are sandwich assays which use two sets of MAB specific for PSA: (1 ) one set of MAB ("capture antibodies") is bound onto a solid phase to capture PSA in a sample, and (2) the other set of MAB ("probe antibodies") is labelled and in free solution to bind the captured PSA for its detection. These assays are herein referred to as "MONO assays". Generally, the binding of these MAB to PSA is not prevented by the binding of ACT to PSA. That is, generally, these MAB can bind both free PSA and the PSA-ACT complex. Examples of such assays are the Hybritech Tandem-E and Tandem-R PSA Assays (Hybritech, La Jolla, Calif.).
The second type of sandwich assays uses MAB specific for PSA on the solid phase, and polyclonal antibodies to PSA as probe antibodies. Generally, in these assays, the MAB can bind both free PSA and the PSA-ACT complex. In contrast, the pool of polyclonal antibodies contains antibodies which can bind both free PSA and the PSA-ACT complex, and antibodies which can bind free PSA but not the PSA-ACT complex. In the latter case, the epitopes bound by these antibodies are blocked by the binding of the ACT to PSA. Examples of these assays are the Abbott IMx.RTM. PSA Assay (Abbott Laboratories, Abbott Park, Ill.), and the ACS.TM. PSA Assay (Ciba-Corning Diagnostics Corporation, East Walpole, Mass.).
It has been found that the second type of sandwich assays preferentially detects free PSA over that of PSA-ACT complex (this phenomenon is herein referred to as "bias"). On the other hand, some MONO assays do not exhibit such a bias. See Bluestein, B., et al., J. Tumor Marker Oncology, 7(4) 41 (1992).
It has been found that in the serum of patients with benign prostatic hyperplasia (BPH), there are more free PSA than PSA-ACT complexes. On the other hand, in the serum of prostate cancer patients, there are more PSA-ACT complexes than free PSA. (Lilja, H., et al., Clin. Chem., 37:1618 (1991); Stenman, U., et al., Cancer Res., 51:222 ( 1991 ); Lilja, H., et al., Cancer Suppl., 70:230 (1992); Christensson, A., et al., J. Urology, 150: 100 (1993)).